Melusin a muscle specific protein, as a drug target for prevention and treatment of heart failure

ABSTRACT

The present invention concerns non-human transgenic animals as model study for human pathologies, being transgenic for having altered melusin expression. The non-human transgenic animals are to be used as models to study heart pathologies and provide therapies thereof, wherein the heart pathologies are heart failure, and in particular diluted cardiomyopathy.

This application is the U.S. national phase of international applicationPCT/IT2002/000807 filed 19 Dec. 2002 which designated the U.S., theentire content of which is hereby incorporated by reference.

FIELD OF INVENTION

The present invention concerns non-human transgenic animals as modelstudy for human pathologies, being transgenic for having altered melusinexpression. The non-human transgenic animals are to be used as models tostudy heart pathologies and provide therapies thereof, wherein the heartpathologies are heart failure, and in particular dilated cardiomyopathy.

In a further embodiment the present invention concerns methods for thedevelopment of therapeutical approaches for prevention and treatment ofhearth failure, in particular dilated cardiomiopathy, by means of usingmelusin protein and/or nucleic acids encoding for melusin protein,fragments and/or derivatives thereof.

BACKGROUND OF INVENTION

In subjects affected by arterial hypertension, the left ventricle of theheart is subjected to increased mechanical activity in order to pumpblood against increased blood pressure. Under these conditions the heartundergoes a compensatory hypertrophy in which cardiomyocytes increase insize as consequence of increased synthesis and assembly of contractileproteins of actomyosin fibrils.

Although hypertrophy is compensatory and beneficial allowing thegeneration of more contractile force, under condition of chronic highblood pressure, additional events might occur that either reduce theefficacy of the hypertrophy response or activate additional pathwayscausing cardiac dilation and progressively leading to heart dysfunctionand failure.

The identification of the molecular mechanisms involved in the initialcardiac hypertrophy and in the onset of a subsequent defectivecardiomyocytes response is a major challenge of the cardiovascularbiology and medicine in these days. In fact, understanding suchmolecular mechanisms can be of great importance to develop therapeuticalstrategies aimed to fight congestive heart failure, a pathology that,only in the United States of America, affects more that 400.000 peoplesevery year.

Considerable efforts have been made in the past decade to identify themolecular mechanisms at the cellular level involved in the hypertrophicresponse of cardiomyocytes. These studies led to a mechanistic modelillustrated in FIG. 10 in which mechanical stretching induced byhemodynamic overload (1) trigger intracellular mechanosensors (2) thatactivate intracellular signaling pathways (3) leading to hypertrophy bydual modes: by direct activation of muscle specific genes (4a) and byinducing secretion of neurohumoral and autocrine factors (4b) that inturn act on the cardiomyocytes via specific receptors and signalingcontributing to the hypertrophic response.

Among the signaling molecules (point 3 of FIG. 10) thought to beinvolved in the cardiac hypertrophy in response to mechanical overloadare: the alfa Gq subunit of the heterotrimeric G protein coupled to thebeta adrenergic receptors (Akhter et al. 1998), the phospholipase C betaand protein kinase C, acting downstream of the G proteins (Wakasaki etal. 1997), the Calcineurin/NF-AT3 pathway, the Ras cascade includingRaf-1 and ERK1/2 MAP kinases, the stress kinases Jnk and p38, thephosphoinositide 3-kinase, the Jak-STAT pathway (for review see Aoki andIzumo 2001; Ruwhof and van der Laarse 2000; Hunter and Chien 1999).These molecules, although very important in inducing the hypertrophicresponse, are all acting quite downstream along the signaling pathways.

It is thus clear that identification of the mechanosensors themselves(point 2 of FIG. 10) would be of great importance, since interferencewith such upstream regulatory elements would allow a much more specificcontrol of the hypertrophic response.

The mechanical tension in the muscle is exerted by the contractileproteins of the cytoskeleton, the actomyosin fibrils which arephysically linked to the plasma membrane and to the extracellularmatrix, via membrane receptors belonging to the integrin family.

In muscles, integrins are preferentially localized in specific sitesknown as myotendinous junction and costamers. These are specific siteswere actomyosin fibrils are connected to the plasma membranecontributing to a correct and stable association of the contractilemachinery to the membrane of the muscle cells.

Besides transmitting the contractile force across the plasma membrane,these junctions are also important mechanosensors capable oftransmitting signals inside the cell in response to mechanicalstretching. Several proteins are in fact localized at these sites at thecytoplasmic face of the plasma membrane and interacting with integrins.These proteins include paxillin, vinculin, talin, and the tyrosinekinase p125Fak. This molecular machinery is activated by mechanicalstretching of the cells (for review see Davis et al. 2001; Carson andWei 2000) and is the best candidate as the mechanosensing apparatus.

A beta1 integrin isoform (beta1D) that is specifically expressed instriated cardiac and skeletal muscle has been disclosed (Belkin et al1996). In association with the alpha7 subunit, beta1D forms anheterodimer a7b1D with receptor activity toward merosin (laminin 2) ofthe extracellular matrix. Functional analysis indicated that beta1Dintegrin binds both cytoskeletal elements and extracellular matrixligands with much higher affinity compared to the beta1A isoform presentin all non-muscle tissues (Belkin et al 1997) suggesting that beta1Dprovides a stable actin-laminin interaction across the plasma membranenecessary to support the mechanical tension during muscle contraction.

To further define the molecular basis of these functional properties theinventors searched for proteins capable to bind to the cytoplasmicdomain of beta1D. Using the two-hybrid screening the inventors isolatedmelusin, a novel protein selectively expressed in skeletal muscle andheart (Brancaccio et al. 1999; GenBank AF140690; GenBank AF140691).

Sequence analysis of melusin indicated the presence in the aminoterminal half of the protein of a tandem repeated cysteine and histidinerich sequence and of putative binding sites for SH2 and SH3 domains. TheC terminal half comprises the binding site for the integrin cytoplasmicdomain and is characterized by a stretch of acidic amino acid residuesbinding to Ca²⁺ (FIG. 1). Melusin is localized at costamers incorrespondence of Z line where also integrins and vinculin areconcentrated (Brancaccio et al. 1999).

Melusin, thus likely represents a new intracellular transducer of beta1integrin function in muscle cells.

DESCRIPTION OF THE INVENTION

The invention concerns non-human transgenic animals as model study forhuman pathologies, being transgenic for having altered melusinexpression. Preferably the human pathology is included in the followinggroup: heart failure, dilated cardiomyopathy, hypertensivecardiomyopathy, hypertrophic cardiomyopathy, congestive heart failure,heart infarct. More preferably the human pathology is dilatedcardiomyopathy.

In a preferred embodiment the non-human transgenic animal subjected toexperimental hypertension conditions, such as for example surgicalconstriction of the aorta, pharmacological treatment with hypertensivedrugs or high sodium diet, exhibits dilated cardiac hypertrophy andcontractile dysfunction and is useful as a study model to providetherapies thereof.

It is a further object of the invention cells derivable from thenon-human transgenic animal of the invention. The invention concernsdifferent uses of the cells for the selection of moleculespharmacologically effective in triggering melusin activation.

Another aspect of the invention relates to a method for the preparationof a non-human transgenic animal comprising essentially the steps of i)preparing a non-human transgenic animal carrying an inactivated melusinallele; ii) breeding the parent transgenic animal with anothernon-transgenic animal; iii) selecting transgenic animals heterozygotefor the melusin mutation; iv) breeding of the heterozygote animals toselect homozygote animals for the melusin gene mutation.

Another aspect of the invention relates to the preparation of anon-human transgenic animal in which the melusin gene is inactivated bygenetic approaches distinct from homologous recombination, such as forexample antisense RNA or RNA interference approaches in which short RNAsequences complementary to melusin transcript or DNA are used tointerfere with either transcription or translation of the melusin gene.

In a further embodiment the present invention concerns methods for thedevelopment of therapeutical approaches for prevention and treatment ofhearth failure, in particular dilated cardiomiopathy, by means of usingmelusin protein and/or nucleic acids encoding for melusin protein,fragments and/or derivatives thereof.

In a related aspect, the invention provides methods for identifyingchemical compounds that are agonists of melusin, being such agonists inthe form of peptides or structural analog organic compounds, whichconsequently can be used for the manufacture of pharmaceuticalcompositions for the therapy of heart pathologies.

The present invention relates also to the use of melusin for themanufacture of a medicament for treatment and prevention of hearthpathologies in humans; in particular relates to the use of i) melusinprotein, peptides, fragments and/or derivatives thereof, ii) nucleicacids encoding for melusin protein, peptides, fragments and/orderivatives thereof for the preparation of pharmaceutical compositionsfor the prevention and treatment of heart pathologies.

A further aspect of the present invention relates to proteins able tointeract with melusin and acting as downstream transducers of themechano-chemical signal and leading to the activation of genes of thehypertrophy cardiomyocyte program. Chemical compounds acting as agonisttoward such proteins can be utilized as potential drug in the preventionand treatment of hear failure.

According to the present invention, these purposes are achieved by meansof the claims which follow.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in detail with reference tothe attached drawings, which are provided purely by way of non-limitingexamples and in which:

FIG. 1. Amino acid sequence of Mouse (Mo) and Human (Hu) melusins asdeduced from the corresponding cDNAs (murine GenBank AF140691, SEQ IDNO: 1; human GenBank AF140690, SEQ ID NO: 2). Underlined are thecysteine and histidine rich domains (continuous) and the carboxyterminal acidic domain (dotted). Putative binding sites for SH2 and SH3domains are indicated in bold. The integrin-binding region is boxed.Vertical bars indicate identical amino acids between mouse and humanmolecules, while double dots are conserved residues. (Brancaccio et al.1999).

FIG. 2. Genomic DNA construct utilized for the homologous recombinationevent in ES cells.

FIG. 3. Southern Blot analysis of the ES cells carrying the mutatedmelusin gene.

FIG. 4: Western blot analysis of melusin and integrins expression inwild type and mutant mice.

FIG. 5. Echocardiographic and hemodynamic parameters in wild type (WT)and melusin-null (KO) hearts in basal conditions. FIG. 5A, leftventricular end diastolic chamber diameter (LVEDD), left ventricular endsystolic chamber diameter (LVESD), interventricular septum thickness inend diastole (IVSD) and left ventricular posterior wall thickness in enddiastole (LVPWD). FIG. 5B left ventricular weight expressed as mg per grof body weight (LVW/BW). FIG. 5C, fractional shortening (FS) of leftventricle calculated as [(LVEDD-LVESD)/LVEDD]×100. FIG. 5D, leftventricular pressure measured in anesthetized mice by a French highfidelity catheter-tip micro manometer (Millar Instrument).

FIG. 6. Scheme of the surgical constriction of the transverse aorta(TAC) utilized to induce pressure overload.

FIG. 7. Left ventricle growth response to transverse aortic constrictionin wild type (+/+) and mutant mice (−/−).

FIG. 8. Echocardiographic parameters in wild type (WT) and melusin-null(KO) hearts of control mice (Sham) or mice subjected to transverseaortic constriction (TAC). FIG. 8A, left ventricular end diastolicchamber diameter (LVEDD). FIG. 8B, left ventricular end systolic chamberdiameter (LVESD). FIG. 8C, interventricular septum thickness in enddiastole (IVSD) and left ventricular posterior wall thickness in enddiastole (LVPWD).

FIG. 9. Left ventricle growth response to sub-pressor doses ofphenylephrine and angiotensin II. Cardiac hypertrophy was evaluated byleft ventricular/body weight ratio as indicated on the Y-axis. −/− and+/+ indicate the mutant (black squares) and wild type (empty squares)mice respectively.

FIG. 10. Diagrammatic representation of the molecular mechanism at thecellular level involved in hypertrophic response of cardiomyocytes.

FIG. 11. Left ventricle remodeling and function after 2 and 4 weeks fromTAC in wild-type (WT, empty bars) and mutant mice (KO, filled bars).FIG. 11 a: Representative M-mode left ventricular echocardiograficrecording of wild-type (upper pictures) and melusin-null (lowerpictures) mice. FIG. 11 b: Left ventricular end diastolic diameter(LVEDD). FIG. 11 c: interventricular septum thickness in end-diastole(IVSTD). FIG. 11 d: percent fractional shortening (% FS) as parameter ofleft ventricle contractile function. FIG. 11 e: Representative grossmorphology of whole hearts (upper rows) and transversal sections at baselevel of the left ventricles of wild-type and melusin-null mice after 4weeks from TAC. §: P<0.01 vs Basal; *: P<0.01 vs wild-type; °: P<0.05 vswild-type.

FIG. 12. Impaired GSK3β phosphorylation in melusin-null mice in responseto TAC by comparison of results between Wild-type (WT, empty bars) andmelusin-null mice (KO, filled bars). Left ventricle protein extractswere analyzed by western blotting with antibodies to phosphorylatedsignaling molecules. Sample loading was controlled using antibodiesspecific for each protein. The intensity of the bands from twoindependent experiments with a total of 8 mice for group was measuredand relative intensity was calculated after subtraction of basal levelin sham operated animals. ERK (FIG. 12 a), p38 (FIG. 12 b), GSK3β (FIG.12 c), AKT (FIG. 12 d) are rapidly phosphorylated in response to TAC inwild-type mice, but phosphorylation of GSK3β and AKT were stronglyimpaired in melusin-null mice. GSK3β was phosphorylated to a comparablelevel in both wild-type and melusin-null mice 10 and 20 min after IPinjection of 2 IU of insulin (FIG. 12 e). Reduced serine9-GSK3βphosphorylation (FIG. 12 f) and increased kinase activity (FIG. 12 g)were detected in melusin-null hearts subjected to 7 days of TAC. °:P<0.05 vs wild-type.

The present invention will now be described in relation to somepreferred embodiments by way of non-limiting examples.

As it will be apparent from the results described below, melusin plays acrucial role in the mechano-chemical signaling leading to a correctcardiomyocytes hypertrophy in response to pressure overload.

In the absence of melusin, cardiac hypertrophy is severely impaired andleft ventricle undergoes dilation, thinning and displays reducedcontractile capacity, thus becoming unable to withstand thebiomechanical stress imposed by the high blood pressure.

Stimulating melusin function and signaling will expectedly improvecardiomyocytes hypertrophy preventing dilation and subsequent heartfailure. Therapeutic strategies will, thus, involve expression orover-expression of melusin in failing hearts by gene transfer, use ofdrugs acting as agonists of melusin or of melusin downstream effectormolecules.

Although several molecules such as the alfa Gq subunit, thephospholipase C beta, the protein kinase C, calcineurin, NF-AT3, Ras,Raf-1, ERK1/2, Jnk and p38MAP kinases, the phosphoinositide kinase 3 andthe STATs (see discussion above) are potential targets for drugs aimedto stimulate heart hypertrophy, all these proteins have the greatdisadvantage of being ubiquitously expressed in most, if not all,tissues. A drug regulating any of these proteins will, thus, unavoidablycause deleterious side effects. Melusin, being a muscle specificprotein, will not present this problem and, on the contrary, representsan ideal target molecule for such type of drugs. A second importantadvantage of melusin over other proteins involved in the hearthypertrophy is its role as mechanosensor, which places melusin in thevery early steps of the biochemical signaling cascade triggering thehypertrophic response. Thus, regulating melusin function allows a veryspecific control of the heart response.

The non-human transgenic animals in which the melusin gene isinactivated by homologous recombination or by genetic approachesdifferent from homologous recombination represent a unique animal modelfor testing drugs aimed to prevent heart failure. In fact, these animalsdo not show functional heart defects in basal conditions during theirlifespan. Heart failure become apparent only when transgenic animals andmore preferably melusin-null transgenic mice are exposed to chronichypertensive conditions. In a preferred embodiment the hypertensiveconditions are determined by surgical constriction of the aorta,pharmacological treatment with hypertensive drugs or high sodium diet.In these conditions, the transgenic mice develop heart dilation andfailure with a relatively slow kinetics (within 4 weeks), a time coursemuch slower compared to that shown by other animal models for dilatedcardiomyopathy (Arbet et al 1997; Hirota et al 1999; Badorff et al 2002)allowing to more accurately test drugs aimed to prevent cardiac failure.

The preparation of a non-human transgenic animal—preferably melusin-nulltransgenic animal—comprises essentially the steps of: i) preparing agenomic DNA construct abrogating melusin expression and suitable forhomologous recombination event; ii) use of such DNA construct to inducehomologous recombination in embryonic stem cells; iii) use of stem cellscarrying an inactivated melusin gene to generate a chimeric embryo; iv)selecting animals heterozygote and homozygote for the melusin mutationby breeding the chimeric animals with different mouse strains.

A transgenic animal for melusin is an animal in which the expression ofthe melusin protein has been altered/modified either in a positive ornegative direction by stable or transient introduction in some or allcells of the animals of molecules capable to modify melusin expressionat transcriptional, translational or post-translational level.

As a non limiting example a DNA construct coding for an antisensemelusin transcript can be used. Expression of such DNA construct isdirected by a cardiac specific promoter such as the promoter of thea-myosin heavy chain. This construct is introduced in fertilized oocytesthat are then reimplanted in the uterus of foster mothers in order togenerate non human transgenic animals that express either none orreduced level of melusin in heart. A second non limiting exampleconsists in the use of vectors coding short duplex RNAs of 21-23 ntcapable to silence genes containing homologous sequences (Hasuwa et al.,2002).

The cardiac pathology displayed by the melusin-null transgenic micedescribed in the present invention indicates that melusin is required tosustain the compensatory hypertrophic response when the heart is exposedto pressure overload. It is thus concluded that the inhibition ofmelusin function by natural or synthetic compounds can lead to cardiacfailure and dilation in animals carrying wild type melusin genes andexposed to hypertensive conditions.

EXAMPLES Example 1 Production of Melusin-Null Trangenic Mice andMolecular Characterization

To investigate the role of melusin in integrin function the inventorshave generated a mutation in mice (inbred 129SV strain) that abrogatemelusin expression. Using the murine cDNA (Brancaccio et al. 1999;GenBank AF140691) the present inventors isolated a genomic fragment of14.8 Kb encompassing four exons at the 5′ end of the melusin gene.Partial characterization by restriction map and sequencing indicatedthat the first exon contains the ATG start codon. A PstI fragmentcontaining exons 1 to 4 was replaced with a cassette containing IRESsequences linked to the LacZ gene followed by the neomycin resistancegene driven by a PGK promoter (FIG. 2). This construct, which has twoarms of 4.1 and 5 Kb homologous to the endogenous gene, waselectroporated in embryonic stem R1 cells from male 129SV inbred mice.Different clones in which homologous recombination occurred have beenidentified by Southern blot analysis (FIG. 3). Since the melusin gene islocated on the X chromosome (Brancaccio et al 1999) and the ES R1 cellsare of male origin, a single homologous recombination event wassufficient to inactivate the melusin gene (FIG. 3). After injection ofthe mutant ES cells into blastocysts and implant in the uterus of afoster mother, chimeric mice in which the genetically modified cellshave colonized the germ line were obtained. Chimeras were than breedwith 129SV mice to obtain melusin-null 129SV mice.

The melusin-null mice are viable and fertile and do not show appreciablemuscle or heart defects up to 18 months of age.

The successful inactivation of the melusin gene was demonstrated byanalysis of protein expression both in heart and skeletal muscles ofmutant mice (FIG. 4). These data indicate, thus, that melusin is notrequired for muscle and heart development.

Example 2 Role of Melusin in Cardiac Hypertrophy

Basal cardiac morphology and performance was investigated byechocardiography and cardiac catheterization and found to be comparablein wild type and melusin-null mice.

As shown in FIG. 5A echocardiography allowed to measure left ventricleend-diastolic (LVEDD), end-systolic diameter (LVESD), interventricularseptum thickness in end diastole (IVSD), left ventricle posterior wallthickness in end diastole (LVPWD) and fractional shortening (FS) (FIG.5C). Left ventricle mass (LVW/BW) was also measured as hypertrophyparameter and found to be comparable in melusin-null and control mice(FIG. 5B). In addition the pressure developed by the left ventricle wasdirectly measured by catheterization with a micro manometer and reportedas dP/dt (FIG. 5D). Thus the absence of melusin does not affect cardiacfunction under physiological conditions.

Melusin deficiency, however, affects cardiac muscle function when heartsare exposed to pressure overload. To analyze the ability to respond tobiomechanical overload, melusin-null hearts were subjected to chronichypertension realized by surgical constriction of the transverse aortaas described below and in FIG. 6.

Mice were anesthetized by injection of a mixture of ketamine (100 mg/kg)and xylazine (10 mg/kg). After midline sthernotomy, the aortic arch isconstricted between truncus anonimus and left carotid artery with 8-0silk tied against the vessel and a blunted 27-gauge needle, which ispromptly pulled out thereafter. In the control group the same surgicalprocedures are performed without constricting the aortic arch. Both wildtype and melusin-null mice were subjected to the above surgicalprocedure. 7 days after surgery the degree of hemodynamic overload wasevaluated as systolic pressure gradient measured by selectivecannulation of left and right carotid arteries (Lembo et al. 1996)

After these hemodynamic evaluations, the mice are weighed, heartsexcised and evaluation of cardiac hypertrophy obtained with leftventricular weight/body weight ratio.

While wild type mice develop an overt compensatory cardiac hypertrophy 7days after surgery, melusin-null mice show only a very modesthypertrophy, evaluated by left ventricular/body weight ratio (FIG. 7).

To better characterize the evolution of the left ventricle remodelingduring chronic pressure overload, melusin-null mice were subjected toTAC (transverse aortic constriction) for a period of 4 weeks andexamined by serial echocardiographic analysis during this period.Cardiac structure and function were evaluated not invasively withtransthoracic echocardiography in basal condition and after 2 and 4weeks from TAC. All measurements were determined in a short axis view atthe level of papillary muscles.

As expected, wild-type mice showed increased interventricular septumthickness and reduced end-distolic left ventricular diameters. Incontrast, after 7 days of TAC, melusin-null mice developed only modestthickening of ventricular walls and a significant chamber enlargement(FIG. 8). After 2 weeks from TAC, melusin-null mice showed a furtherenlargement of left ventricular chamber as compared to that observed inwild-type mice (FIG. 11). After 4 weeks, left ventricular dilation waseven more evident and associated with a marked deterioration ofcontractile function, as detected by the severe impairment of fractionalshortening (FIG. 11). Finally, the lethality rates at 4 weeks from TACwere greater in mutant as compared to wild-type mice (53.3% vs 30.7%).

The absence of melusin results, thus, in reduced cardiac hypertrophy andpromotes the left ventricle dilation when hearts are exposed toincreased blood pressure. This condition is, thus, accelerating theonset of the defective cardiac response.

Example 3 Mechanosensor Role of Melusin in the Heart

To test whether melusin is involved in heart hypertrophy in response tostimuli different from pressure overload, the inventors also tested thecardiac response in melusin-null mice after chronic administration ofphenylephrine or angiotensin II at sub-pressor doses which do notincrease blood pressure.

Chronic administration of sub-pressor doses of phenylephrine (100mg/kg/day) or angiotensin II (0,1 mg/kg/day) (Harada et al, 1998) wasobtained by subcutaneous implantation of osmotic mini-pump (Alza Corp.)delivering the above doses of phenylephrine or angiotensin II for 21days. In these experimental series control groups were treated withvehicle alone.

To verify that chronic agonists infusion does not alter blood pressurehomeostasis, blood pressure profile was evaluated by radio-telemetricmeasurement realized through implantation of a commercially availabledevice into the femoral artery and acquisition of the telemeteredpressure signal in a dedicated, computed analysis system (Data SciencesInternational).

Cardiac hypertrophy was evaluated by left ventricular/body weight ratio.The results of these experiments indicate that melusin-null mice exposedto sub-pressor doses of phenylephrine or angiotensin II develop leftventricle hypertrophy in a manner not significantly different from wildtype mice (FIG. 9).

These results altogether indicate that melusin is involved in thehypertrophic response to pressure overload (see Example 2), but is notrequired in the response to trophic factors such as phenylephrine orangiotensin II. This, thus, strongly points for a mechanosensor role ofmelusin in heart.

Example 4 The Lack of Melusin Impairs GSK3β Phosphorylation

To investigate the impact of melusin on cardiac intracellular signalingtriggered by biomechanical stress, phosphorylation of signaling proteinsreported to be involved in cardiac hypertrophy (Aoki and Izumo 2001;Hunter and Chien 1999; Hardt and Sadoshima, Circ Res. 2002) wasanalyzed.

Representative experiments are shown in FIG. 12. Wild-type (WT, emptybars) and melusin-null mice (KO, filled bars) were subjected to TAC for10 min or to sham (S) operations as controls. Left ventricle proteinextracts were analyzed by western blotting with antibodies tophosphorylated signaling molecules.

In particular the inventors found that glycogen synthase kinase 3beta(GSK3β) was differentially phosphorylated in wild type versusmelusin-null mice. As shown in FIG. 12 c, GSK3β was stronglyphosphorylated at serine 9 residue 10 minutes after TAC in wild-typemice consistently with the hypothesis that this signal was triggered inresponse to a mechanical event. However, in melusin-null mice the degreeof GSK3β serine 9 phosphorylation was severely reduced (FIG. 12 c).

Since AKT is a major kinase regulating GSK3β serine 9 phosphorylationthe inventors analyzed the phosphorylation state of this kinase. WhileAKT is rapidly phosphorylated in response to TAC in wild type mice, thisresponse was strongly reduced in melusin-null mice (FIG. 12 d).

GSK3β is involved in multiple signaling pathways and is a well knowntarget of insulin receptor signaling (Cohen and Frame 2001). Theinventors then tested whether the lack of melusin could affect GSK3βphosphorylation in response to insulin. Western blot analysis of heartextracts from mice treated for 10 and 20 min with insulin—administeredwith IP injections of 2IU—showed that GSK3β was phosphorylated atcomparable level in both mice genotypes (FIG. 12 e).

Since attenuation of cardiac hypertrophy in melusin-null mice wasobserved after 7 day TAC, the inventors then tested GSK3β signaling atthis point in time. Interestingly GSK3β serine 9 phosphorylation wasreduced in melusin-null mice versus wild type after 7 days of TAC (FIG.12 f). In addition kinase activity was increased as predicted by theinhibitory action of serine 9 phosphorylation (FIG. 12 g). Thus alteredGSK3β signaling is persistent in melusin-null mice exposed to 7 day TAC.

These data indicate that the lack of melusin selectively impairs leftventricular AKT and GSK3β phosphorylation in response to biomechanicalstress.

Example 5 Isolation of Melusin-Agonist Organic Compound

In order to identify melusin agonists, molecules capable of activatingmelusin function have to be identified. Based on the previouslypublished (Brancaccio et al 1999) and present data, integrins bindmelusin and trigger its activation. Thus, peptides or organic compoundsbinding to melusin and interfering with melusin-integrin binding areexpectedly good candidates as melusin agonists by mimicking theintegrin-induced melusin activation. Such melusin agonists can beidentified from a large library of peptide and peptide-like compounds byusing the techniques of high-throughput screening using a well-definedassay for the detection of such agonists

To this end, an ELISA assay can be used in which purified recombinantmelusin, or fragments of the protein is adsorbed on the surface ofmicrotiter wells are incubated with cell extracts containing integrin toallow integrin-melusin binding. To this mixture specific compounds areadded to select molecules capable to bind melusin and prevent integrinbinding. A similar procedure has already been used to select compoundcapable to interfere with integrin function in other cellular systems(Ambroise et al. 2002)

The following ELISA protocol can be used. Fusion proteins consisting GST(Glutathione S-trasferase) fused to the full-length human melusin and/ora fragment from amino acid residues 149-350 in the C-terminal region,containing the integrin binding site (Brancaccio et al 1999) arepurified by affinity chromatography on glutathione-Sepharose 4B.Purified fusion proteins are adsorbed on microtiter wells according tostandard procedures and used as ligands for integrin binding. COS cellextracts are used as source of beta1 integrin heterodimers.

Briefly COS cells are washed twice with cold PBS and extracted in TBS(25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM NaVO4, 10 mM NaF, 10 μg/mlleupeptin, 4 μg/ml pepstatin and 0.1 TIU/ml aprotinin) 0.5% Nonidet P-40plus 1 mM Ca²⁺, or with TBS, 0.5% Nonidet P-40 plus 5 mM EDTA tosolubilized membrane proteins. 100 μl of cell extracts containing 2 mgof proteins/ml are incubated overnight at 4° C. in wells coated withGST-melusin, GST-melusin(aa149-350) and GST alone (as control). Afterwashing, integrin binding is detected with the TS2/16 monoclonalantibody followed by peroxidase-conjugated anti-mouse antibody. Thecompounds capable to interfere with integrin-melusin binding will beadded at increasing concentrations together with the COS cell extractduring the incubation with the GST-melusin fusion protein.

As source of compounds capable to interfere with melusin-integrinbinding, random peptides phage display library can be used (Ladner andLey 2001). In such libraries random oligonucleotide sequences coding forshort amino acid sequences (8-18 residues) are inserted in the codingsequence of the phage coat proteins. The resulting phage displays on itssurface the random peptide sequence to be selected for its bindingcapacity. The phage population displaying the random peptides is allowedto interact with recombinant melusin adsorbed on the surface ofmicrotiter wells before incubation with integrins. Phages interferingwith integrin binding are isolated and the peptide sequence coded for bythe inserted random oligonucleotide will be determined by DNAsequencing.

As an alternative source of organic compounds capable to bind melusininterfering with integrin binding combinatorial chemistry libraries areused (Floyd et al. 1999; Ambroise et al. 2002; Toogood 2002).

The peptide sequences isolated with the procedure described above willbe tested for their ability to trigger in vitro cardiomyocytehypertrophy. For this test melusin-null cardiomyocytes derived from thetrangenic animals of the invention are used to define the specificity ofthe isolated compound toward melusin. Such compound are ineffective onmelusin-null cardiomycytes while they should be active on wild typecells.

To allow penetration in the cells, the peptides are coupled to trojanpeptides (Derossi et al 1998) that allow spontaneous and efficientintracellular delivery.

Active peptides are also used to develop structural analog organiccompounds more suitable for in vivo treatment.

Genetically modified mice models developing, either spontaneous orpressure overload-induced, dilated cardiomyopathy (for review see Chien,1999) are treated by delivering peptide analogs with subcutaneousimplantation of infusion mini-pumps and analyzed for their cardiacfunction and morphology to monitor the in vivo potential therapeuticactivity of the compounds.

The same strategy is applied with molecules acting downstream of melusinin controlling the cardiac hypertrophy response. Using differentexperimental approaches, including co-immunoprecipitation, affinitychromatography and the two hybrid test, proteins binding to melusin andfunctioning as downstream transducers of the mechanochemical signalleading to hypertrophy can be identified (between step 2 and 3 in FIG.10). Once these molecules are identified and characterized, the samestrategy described above can be applied to select agonists that boostthe activation of such proteins acting downstream of melusin. Such drugsare tested in melusin-null transgenic mice for their ability to rescueleft ventricle dilation observed in these animals after TAC.

Example 6 Melusin Gene-Therapy to Prevent and Treat Heart Dilation andFailure

As discussed above an alternative therapeutic strategy to prevent and/orcure heart dilation and failure can be achieved by inducingover-expression of melusin.

Adenovirus constructs and/or other viral vectors such as lentiviralvectors have been proved efficient vector for gene delivery inexperimental heart pathologies (Wright et al 2001). Adenoviral vectorexpressing the melusin gene are prepared according to the followingprotocol given as an example. Lentiviral vectors can be prepared bysimilar procedures as well.

Human melusin cDNA (GenBank AF140690) is cloned in a shuttle vector(pAdTrack-CMV) containing GFP marker. 100-500 ng of the resultantplasmid is linearized by digestion with PmeI restriction endonuclease,and after digestion extracted with phenol-cloroform treatment,precipitated with etanol and resuspended in 6 μl of deionised water.PmeI-digested shuttle plasmid is co-transformed with 100 ng ofadenoviral backbone vector (pAdEasy-1) by electroporation in BJ5183 E.coli cells. Transformed E. coli cells are resuspended in 500 μl ofL-broth, plated on 3 LB kanamycin plates and grown overnight at 37° C.10-20 colonies are picked up and grown in 2 ml L-broth containing 25μg/ml kanamycin for 10-15 hours. DNA minipreps are performed withconventional methods and supercoiled plasmids are digested with PacIrestriction endonuclease. Candidate clones yield after digestion a largefragment of about 30 kb and a smaller one of 3 or 4.5 kb. Recombinantadenovirus is retransformed in E. coli and is purified usingcommercially available purifications kits. 4 μg of recombinantadenoviral DNA is then linearized wth PacI restriction endonuclease,precipitated with ethanol, resuspended in 20 μl of sterile water andused for transfection of 2×10⁶ 293 cells (E1-transformed human embryonickidney cells) at 50-70% confluence. Transfection and viral production ismonitored by the fluorescent protein GFP expression. Cells are scrapedwith a rubber policeman at 7-10 days post-transfection and collected in50 ml tubes, then spinned in a centrifuge and resuspended in 2 mlsterile PBS. Cells are frozen in dry ice-methanol bath and are thawed ina 37° C. water bath and then vortexed. Cells are frozen and thawed for atotal of 4 cycles. Then samples are spinned briefly and stored at −20°C. This viral supernatant is used to infect 50-70% confluent 293 cellsin order to produce large amount of viral stocks. The virus arecollected when a third to half of the cells are detached. It is possibleto confirm the virus presence using PCR and western blot analysis. Viraltiter is measured by counting green fluorescent 293 cells 18 hours afterinfection with various dilutions of virus supernatant.

The adenoviral vector is delivered to animals following a catheter-basedprotocol for intracardiac injection according to Hajjar et al. (Hajjaret al 1998).

As proof of efficacy in vivo, the melusin adenoviral vector are testedfor its ability to rescue cardiac dilation in melusin-null micesubjected to transverse aortic constriction. This procedure is alsoapplied on different transgenic mouse models with impaired hypertrophyresponse, or with spontaneous dilated cardiomyopathy.

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1. A transgenic mouse comprising a disruption in its endogenous melusingene, wherein said mouse lacks expression of endogenous melusin, andwherein said mouse, after being subjected to a hypertensive condition,develops at least a phenotype selected from the group consisting ofimpaired heart hypertrophy, heart dilation, and heart failure.
 2. Thetransgenic mouse according to claim 1, wherein said hypertensivecondition is induced by surgical operation.
 3. The transgenic mouseaccording to claim 2, wherein said surgical operation consists ofsurgical constriction of the transverse aorta.
 4. The transgenic mouseaccording to claim 1, wherein said hypertensive condition is induced bypharmacological treatment.
 5. The transgenic mouse according to claim 1,wherein said hypertensive condition is induced by high sodium diet. 6.The transgenic mouse according to claim 1, wherein said mouse developsat least impaired heart hypertrophy.
 7. The transgenic mouse accordingto claim 1, wherein said mouse develops at least heart dilation.
 8. Thetransgenic mouse according to claim 1, wherein said mouse develops atleast heart failure.
 9. The transgenic mouse according to claim 4,wherein said pharmacological treatment is administration of hypertensivedrugs.
 10. The transgenic mouse according to claim 1, wherein said mousebelongs to the 129SV, C57Bl or 129SVxC57Bl strain.
 11. A method ofselecting a compound that is pharmacologically active in the preventionof heart failure, said method comprising: i) administering compounds tothe transgenic mouse according to claim 1, ii) inducing a hypertensivecondition in said mouse, and iii) selecting a compound that ispharmacologically active in the prevention of heart failure.
 12. Amethod of studying a heart pathology, said method comprising: i)exposing the transgenic mouse according to claim 1 to hypertensiveconditions and ii) studying development of a heart pathology in saidmouse, wherein said heart pathology is selected from the groupconsisting of heart failure, congestive heart failure, dilatedcardiomyopathy, hypertensive cardiomyopathy, hypertrophiccardiomyopathy, and heart infarct.
 13. Cells obtained from thetransgenic mouse according to claim
 1. 14. A method of selecting acompound that is pharmacologically active in the prevention of heartfailure, said method comprising: i) administering compounds to the cellsaccording to claim 13, ii) inducing a hypertensive condition in saidcells, and iii) selecting a compound that is pharmacologically active inthe prevention of heart failure.
 15. A method of producing a transgenicmouse comprising a disruption in its endogenous melusin gene, whereinsaid mouse lacks expression of endogenous melusin, and wherein saidmouse after being subjected to a hypertensive condition, develops atleast a phenotype selected from the group consisting of impaired hearthypertrophy, heart dilation, and heart failure, said method comprising:(a) disrupting by homologous recombination the gene encoding melusin ina mouse embryonic stem (ES) cell, (b) injecting said ES cell into amouse blastocyst, (c) implanting said blastocyst into the uterus of afoster mother mouse to generate a chimeric embryo, (d) obtaining achimeric mouse which has germ line cells comprising a disrupted geneencoding melusin from said chimeric embryo, (e) breeding said chimericmouse with a different mouse strain, and (f) selecting a male transgenicmouse comprising disruption of the gene encoding melusin.
 16. The methodaccording to claim 15, further comprising breeding said male transgenicmouse with a female transgenic mouse comprising a heterozygous orhomozygous disruption in its endogenous melusin gene, and selecting ahomozygous female mouse comprising disrupted genes encoding melusin. 17.A method of selecting a compound that is pharmacologically active in thetreatment of heart failure, said method comprising: i) inducing ahypertensive condition in the transgenic mouse according to claim 1, ii)administering compounds to said mouse, and iii) selecting a compoundthat is pharmacologically active in the treatment of heart failure. 18.A method of selecting a compound that is pharmacologically active in thetreatment of heart failure, said method comprising: i) inducing ahypertensive condition in the cells according to claim 13, ii)administering compounds to the said cells, and iii) selecting a compoundthat is pharmacologically active in the treatment of heart failure.